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Abstract:

The invention relates to a method for stimulating the growth of the
plants and/or improving the biomass production and/or increasing the
carbon fixation by the plant comprising introducing into a plant cell,
plant tissue or plant one or more nucleic acids, wherein the introduction
of the nucleic acid(s) results inside the chloroplast of a de novo
expression of one or more polypeptides having the enzymatic activity of a
glycolate dehydrogenase made up from translationally fused subunits of
bacterial multi-subunit glycolate dehydrogenase enzymes.

Claims:

1. A method for increasing biomass production and/or seed production
and/or carbon fixation in plants comprising introducing into the genome
of a plant cell a nucleic acid encoding a glycolate dehydrogenase
multi-subunit fusion protein, wherein said introduction of said one
nucleic acid results in a de novo expression of one polypeptide having
the enzymatic activity of a glycolate dehydrogenase and wherein said one
polypeptide is localized in chloroplasts of the plant produced.

2. The method of claim 1, wherein said introduction of said one nucleic
acid is done into the nuclear genome of the plant cells, and wherein said
one nucleic acid encodes one polypeptide comprising an amino acid
fragment that targets the polypeptide to the chloroplast.

3. The method of claim 1, wherein said glycolate dehydrogenase
multi-subunit fusion protein is the one consisting of the fusion of
bacterial glycolate dehydrogenase subunits.

4. The method of claim 1, wherein the glycolate dehydrogenase
multi-subunit fusion protein is the one consisting of the fusion of the
three subunits encoded by the E. coli glc operon.

14. A transgenic plant cell according to claim 13, wherein the glycolate
dehydrogenase multi-subunit fusion protein comprises an amino acid
sequence which targets said protein to the chloroplast.

15. A transgenic plant cell according to claim 13 wherein said glycolate
dehydrogenase multi-subunit fusion protein is the one consisting of the
fusion of bacterial glycolate dehydrogenase subunits.

16. A transgenic plant cell according to claim 13 wherein said glycolate
dehydrogenase multi-subunit fusion protein is the one consisting of the
fusion of the three subunits encoded by the E. coli glc operon.

19. A transgenic plant, and part thereof, comprising a transgenic plant
cell according to claim 13.

20. A transgenic plant, and part thereof, according to claim 19 selected
among rice, wheat, barley, potato, rapeseed, tobacco.

21. A transgenic seed, and meal, oil or food obtained thereof, comprising
a transgenic plant cell according to claim 13.

22. A transgenic seed, and meal, oil or food obtained thereof, according
to claim 21 selected from the group consisting of rice, wheat, barley,
rapeseed, and tobacco.

Description:

[0001] Crop productivity is influenced by many factors, among which are,
on the one hand factors influencing the capacity of the plant to produce
biomass (photosynthesis, nutrient and water uptake), and on the other
hand factors influencing the capacity of the plant to resist certain
stresses, like biotic stresses (insects, fungi, viruses . . . ) or
abiotic stresses (drought, salinity, nutrient starvation . . . ).

[0002] One important factor influencing the production of biomass is
photosynthesis. Photosynthesis is the mechanism through which plants
capture atmospheric carbon dioxide and transform it into sugar, which is
then incorporated into plant tissues, thereby creating biomass.
Photosynthesis is the ultimate source of all primary productivity on
earth.

[0003] Most plants have a photosynthetic mechanism in which the
chloroplastic enzyme RuBisCo (Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase) is the main enzyme capturing carbon dioxide and
transforming it into sugar. Those plants, including some of the most
important crop plants, e.g. rice, wheat, barley, potato, rapeseed, belong
to the so-called C3 plants. One known problem in the photosynthetic
mechanism of C3 plants is that the efficiency of carbon fixation is not
optimal in certain environmental conditions where part of the fixed
carbon is lost through the alternative activity of RuBisCo called
oxygenation.

RuBisCO is able to catalyze both the carboxylation and oxygenation of
ribulose-1,5-bisphosphate. The balance between these two activities
depends mainly on the CO2/O2 ratio in the leaves, which may
change following the plant's reaction to certain environmental
conditions. Each carboxylation reaction produces two molecules of
phosphoglycerate that enter the Calvin cycle, ultimately to form starch
and sucrose and to regenerate ribulose-1,5-bisphosphate. The oxygenation
reaction produces single molecules of phosphoglycerate and
phosphoglycolate. The latter is recycled into phosphoglycerate by
photorespiration (Leegood R. C. et al, 1995). One molecule of CO2 is
released for every two molecules of phosphoglycolate produced, resulting
in a net loss of fixed carbon that ultimately reduces the production of
sugars and biomass. Ammonia is also lost in this reaction, and needs to
be refixed through energy consuming reactions in the chloroplast.

[0004] Overcoming photorespiration has been reported as a target for
raising the maximum efficiency of photosynthesis and enhancing its
productivity (Zhu et al., 2008) and several attempts have been described
so far to reduce the loss of carbon in plants and therefore to increase
the production of sugars and biomass.

[0005] Kebeish et al. reported that the photorespiratory losses in
Arabidopsis thaliana can be alleviated by introducing into chloroplasts a
bacterial pathway for the catabolism of the photorespiratory
intermediate, glycolate (WO 03/100066; Kebeish R. et al., 2007). The
authors first targeted the three subunits of Escherichia coli glycolate
dehydrogenase to Arabidopsis thaliana chloroplasts and then introduced
the Escherichia coli glyoxylate carboligase and Escherichia coli
tartronic semialdehyde reductase to complete the pathway that converts
glycolate to glycerate in parallel with the endogenous photorespiratory
pathway. This step-wise nuclear transformation with the five Escherichia
coli genes leads to Arabidopsis plants in which chloroplastic glycolate
is converted directly to glycerate. These transgenic plants grew faster,
produced more shoot and root biomass, and contained more soluble sugars.

[0006] In PCT/EP2009/059843, a method for increasing biomass production
and/or seed production and/or carbon fixation in rice plants is
disclosed, wherein the rice plant is transformed with the three subunits
(glcD, glcE and glcF) of Escherichia coli glycolate dehydrogenase,
without subsequent introduction of the Escherichia coli glyoxylate
carboligase and Escherichia coli tartronic semialdehyde reductase.

[0007] The objective of the present invention is to exploit translational
fusions of the subunits bacterial multi-subunit glycolate dehydrogenase
(GDH) enzymes in crops avoiding the time-consuming and cumbersome process
of multiple transformations or of transformation with multiple expression
cassettes. The bacterial glcD, glcE and glcF subunits have been fused
with flexible linkers in different arrangements and tested in E. coli
strains deficient in GDH demonstrating that the recombinant GDH
multi-subunit fusion proteins DEFp, EFDp and FDEp are active. Best
performing constructs have been transferred to Nicotiana tabacum, rice
and rapeseed plants and transgenic plants showed significant increased
growth and improved photosynthetic rate.

[0008] The present invention relates to a method for increasing biomass
production and/or seed production and/or carbon fixation in plants
comprising introducing into the genome of a plant cell a nucleic acid
encoding a glycolate dehydrogenase multi-subunit fusion protein, wherein
said introduction of said one nucleic acid results in a de novo
expression of one synthetic polypeptide having the enzymatic activity of
a glycolate dehydrogenase and wherein said one polypeptide is localized
in chloroplasts of the plant produced.

[0009] In the context of the invention, a glycolate dehydrogenase
multi-subunit fusion protein is one polypeptide consisting of the
subunits of a glycolate dehydrogenase that are essential for glycolate
dehydrogenase activity, generally with peptide linkers in between these
subunits.

[0010] In the present invention we selected the repetitive linker sequence
(Gly4Ser)3 suited to connect covalently the bacterial glcD,
glcE and glcF domains into a polyprotein format without interfering with
the desired properties such as proper folding, solubility and GDH
activity. Furthermore, the linker should not be subjected to the
proteases cleavage in the plant cytosol, allowing the polyprotein
overexpression in chloroplasts.

[0011] In the context of the invention, biomass is the quantity of matter
produced by individual plants, or by surface area on which the plants are
grown. Several parameters may be measured in order to determine the
increase of biomass production. Examples of such parameters are the
height of the plant, surface of the leave blade, shoot dry weight, root
dry weight, seed number, seed weight, seed size . . . . Seed production
or seed yield can be measured per individual plant or per surface area
where the plants are grown.

These parameters are generally measured after a determined period of
growth in soil or at a specific step of growth, for example at the end of
the vegetative period, and compared between plants transformed with the
one or more nucleic acids according to the invention and plants not
transformed with such one or more nucleic acids.

[0012] The increase of carbon fixation by the plant can be determined by
measuring gas exchange and chlorophyll fluorescence parameters. A
convenient methodology, using the LI-6400 system (Li-Cor) and the
software supplied by the manufacturer, is described in R. Kebeish et al.,
2007, and is incorporated herein by reference.

[0013] The nucleic acid involved in the method of the invention encodes
one polypeptide having the enzymatic activity of a glycolate
dehydrogenase.

The glycolate dehydrogenase activity can be assayed according to Lord J.
M. 1972, using the technology described in example 6 of the present
application.

[0014] Alternatively, complementation analysis with mutants of E. coli
deficient in the three subunits forming active endogenous glycolate
dehydrogenase may be performed. These mutants of E. coli are incapable of
growing on glycolate as the sole carbon source. When the overexpression
of an enzyme in these deficient mutants restores the growth of the
bacteria on the medium containing glycolate as the sole carbon source, it
means that this enzyme encodes a functional equivalent to the E. coli
glycolate dehydrogenase. The method and means for the complementation
analysis is described in Bari et al, 2004, and incorporated herein by
reference.

[0015] Nucleic acid molecules encoding one polypeptide having the
enzymatic activity of a glycolate dehydrogenase may be produced by means
of recombinant DNA techniques (e.g. PCR), or by means of chemical
synthesis. The identification and isolation of such nucleic acid
molecules may take place by using the sequences, or part of those
sequences, of the known glycolate dehydrogenases nucleic acid molecules
or, as the case may be, the reverse complement strands of these
molecules, e.g. by hybridization according to standard methods (see e.g.
Sambrook et al., 1989).

[0016] The glycolate dehydrogenase for the purpose of the invention can be
any naturally-occurring glycolate dehydrogenase, or any active fragment
thereof or any variant thereof wherein some amino acids (preferably 1 to
20 amino acids, more preferably 1 to 10, even more preferably 1 to 5)
have been replaced, added or deleted such that the enzyme retains its
glycolate dehydrogenase activity.

[0017] According to the present invention, a "nucleic acid " or "nucleic
acid molecule" is understood as being a polynucleotide molecule which can
be of the DNA or RNA type, preferably of the DNA type, and in particular
double-stranded. It can be of natural or synthetic origin. Synthetic
nucleic acids are generated in vitro. Examples of such synthetic nucleic
acids are those in which the codons which encode polypeptide(s) having
the enzymatic activity of a glycolate dehydrogenase according to the
invention have been optimized in accordance with the host organism in
which it is to be expressed (e.g., by replacing codons with those codons
more preferred or most preferred in codon usage tables of such host
organism or the group to which such host organism belongs, compared to
the original host). Methods for codon optimization are well known to the
skilled person.

[0018] Preferred glycolate dehydrogenase multi-subunit fusion proteins are
those consisting of the fusion of bacterial glycolate dehydrogenase
subunits, more preferably those consisting of the fusion of the three
essential subunits encoded by the E. coli glc operon
(gi/1141710/gb/L43490.1/ECOGLCC). Most preferred are polypeptides which
comprise the fused amino acid sequences of SEQ ID NOs: 2 (Glc D), 4 (Glc
E) and 6 (Glc F), wherein these amino sequences may be linked by a
linker. Accordingly, a nucleic acid comprising the polynucleotide
sequences of SEQ ID NOs: 1, 3 and 5 can be used for performing the
present invention.

[0019] The method of the invention encompasses the introduction into the
genome of a plant cell of a nucleic acid encoding a glycolate
dehydrogenase multi-subunit fusion protein, having the enzymatic activity
of a glycolate dehydrogenase, wherein said polypeptide comprises
sequences having a sequence identity of at least 60, 70, 80 or 90%,
particularly at least 95%, 97%, 98% or at least 99% at the amino acid
sequence level with SEQ ID NO: 2, 4, and 6 respectively, wherein the
introduction of the nucleic acid(s) result in a de novo expression of one
polypeptide having the enzymatic activity of a glycolate dehydrogenase,
and wherein said activity is located inside the chloroplasts.

[0020] The method of the invention encompasses also the introduction into
the genome of a plant cell of a nucleic acid encoding a glycolate
dehydrogenase multi-subunit fusion protein, having the enzymatic activity
of a glycolate dehydrogenase, wherein said nucleic acid comprises nucleic
acid sequences with at least 60, 70, 80 or 90%, particularly at least
95%, 97%, 98% or at least 99%, sequence identity to the nucleotide
sequences of SEQ ID NO: 1, 3, and 5 respectively, wherein the
introduction of the nucleic acid results in a de novo expression of at
least one polypeptide having the enzymatic activity of a glycolate
dehydrogenase, and wherein said activity is located inside the
chloroplasts.

[0021] For the purpose of this invention, the "sequence identity" of two
related nucleotide or amino acid sequences, expressed as a percentage,
refers to the number of positions in the two optimally aligned sequences
which have identical residues (×100) divided by the number of
positions compared. A gap, i.e a position in an alignment where a residue
is present in one sequence but not in the other, is regarded as a
position with non-identical residues. The alignment of the two sequences
can be performed by the Needleman and Wunsch algorithm (Needleman and
Wunsch 1970) in EMBOSS (Rice et al., 2000) to find optimum alignment over
the entire length of the sequences, using default settings (gap opening
penalty 10, gap extension penalty 0.5).

[0022] Once the sequence of a foreign DNA is known, primers and probes can
be developed which specifically recognize these sequences in the nucleic
acid (DNA or RNA) of a sample by way of a molecular biological technique.
For instance, a PCR method can be developed to identify the genes used in
the method of the invention (gdh genes) in biological samples (such as
samples of plants, plant material or products comprising plant material).
Such a PCR is based on at least two specific "primers", e.g., both
recognizing a sequence within the gdh coding region used in the invention
(such as the coding region of SEQ ID No. 1, 3, 5), or one recognizing a
sequence within the gdh coding region and the other recognizing a
sequence within the associated transit peptide sequence or within the
regulatory regions such as the promoter or 3' end of the chimeric gene
comprising a gdh DNA used in the invention. The primers preferably have a
sequence of between 15 and 35 nucleotides which under optimized PCR
conditions specifically recognize a sequence within the gdh chimeric gene
used in the invention, so that a specific fragment ("integration
fragment" or discriminating amplicon) is amplified from a nucleic acid
sample comprising a gdh gene used in the invention. This means that only
the targeted integration fragment, and no other sequence in the plant
genome or foreign DNA, is amplified under optimized PCR conditions.

[0023] The method of the invention encompasses also the introduction into
the genome of a plant cell of a nucleic acid encoding a glycolate
dehydrogenase multi-subunit fusion protein, having the enzymatic activity
of a glycolate dehydrogenase, wherein said one nucleic acid hybridizes
under stringent conditions to a nucleotide sequence selected from the
group of SEQ ID NO 1, 3, and 5, wherein the introduction of the nucleic
acid(s) result in a de novo expression of one polypeptide having the
enzymatic activity of a glycolate dehydrogenase, and wherein said
activity is located inside the chloroplasts. Stringent hybridization
conditions, as used herein, refers particularly to the following
conditions: immobilizing the relevant DNA sequences on a filter, and
prehybridizing the filters for either 1 to 2 hours in 50% formamide, 5%
SSPE, 2×Denhardt's reagent and 0.1% SDS at 42° C., or 1 to 2
hours in 6×SSC, 2×Denhardt's reagent and 0.1% SDS at
68° C. The denatured dig- or radio-labeled probe is then added
directly to the prehybridization fluid and incubation is carried out for
16 to 24 hours at the appropriate temperature mentioned above. After
incubation, the filters are then washed for 30 minutes at room
temperature in 2×SSC, 0.1% SDS, followed by 2 washes of 30 minutes
each at 68° C. in 0.5×SSC and 0.1% SDS. An autoradiograph is
established by exposing the filters for 24 to 48 hours to X-ray film
(Kodak XAR-2 or equivalent) at -70° C. with an intensifying
screen. Of course, equivalent conditions and parameters can be used in
this process while still retaining the desired stringent hybridization
conditions.

[0024] The terminology DNA or protein "comprising" a certain sequence X,
as used throughout the text, refers to a DNA or protein including or
containing at least the sequence X, so that other nucleotide or amino
acid sequences can be included at the 5' (or N-terminal) and/or 3' (or
C-terminal) end, e.g. (the nucleotide sequence encoding) a selectable
marker protein, (the nucleotide sequence encoding) a transit peptide,
and/or a 5' leader sequence or a 3' trailer sequence. Similarly, use of
the term "comprise", "comprising" or "comprises" throughout the text and
the claims of this application should be understood to imply the
inclusion of a stated integer or step or group of integers or steps but
not the exclusion of any other integer or step or group of integers or
steps

[0025] The method of the present invention consists in installing a
glycolate dehydrogenase activity inside the chloroplast. This can be done
either by introducing the nucleic acid encoding the glycolate
dehydrogenase activity into the nuclear genome of plant cells, the coding
sequence of the protein then being fused to a nucleic acid encoding a
chloroplast transit peptide. Alternatively, the glycolate dehydrogenase
activity can be put into the chloroplast by direct transformation of the
chloroplast genome with the nucleic acid(s) encoding the corresponding
enzyme.

[0026] General techniques for transforming plant cells or plants tissues
can be used. One series of methods comprises bombarding cells,
protoplasts or tissues with particles to which the DNA sequences are
attached. Another series of methods comprises using, as the means for
transfer into the plant, a chimeric gene which is inserted into an
Agrobacterium tumefaciens Ti plasmid or an Agrobacterium rhizogenes Ri
plasmid. Other methods may be used, such as microinjection or
electroporation or otherwise direct precipitation using PEG. The skilled
person can select any appropriate method and means for transforming the
plant cell or the plant.

For the purpose of expressing the nucleic acid which encodes the
polypeptide having the enzymatic activity as required for the present
invention in plant cells, any convenient regulatory sequences can be
used. The regulatory sequences will provide transcriptional and
translational initiation as well as termination regions, where the
transcriptional initiation may be constitutive or inducible. The coding
region is operably linked to such regulatory sequences. Suitable
regulatory sequences are represented by the constitutive 35S promoter.
Alternatively, the constitutive ubiquitin promoter can be used, in
particular the maize ubiquitin promoter (GenBank: gi19700915). Examples
for inducible promoters represent the light inducible promoters of the
small subunit of RUBISCO and the promoters of the "light harvesting
complex binding proteins (lhcb)". Advantageously, the promoter region of
the gos2 gene of Oryza sativa including the 5' UTR of the GOS2 gene with
intron (de Pater et al., 1992), the promoter region of the
ribulose-1,5-biphosphate carboxylase small subunit gene of Oryza sativa
(Kyozuka J. et al., 1993), or the promoter region of the actin 1 gene of
Oryza sativa (McElroy D. et al., 1990) may be used.

[0027] According to the invention, use may also be made, in combination
with the promoter, of other regulatory sequences, which are located
between the promoter and the coding sequence, such as transcription
activators ("enhancers"), for instance the translation activator of the
tobacco mosaic virus (TMV) described in Application WO 87/07644, or of
the tobacco etch virus (TEV) described by Carrington & Freed 1990, for
example, or introns such as the adh1 intron of maize or intron 1 of rice
actin.

As a regulatory terminator or polyadenylation sequence, use may be made
of any corresponding sequence of bacterial origin, such as for example
the nos terminator of Agrobacterium tumefaciens, of viral origin, such as
for example the CaMV 35S terminator, or of plant origin, such as for
example a histone terminator as described in Application EP 0 633 317.

[0028] In one particular embodiment of the invention whereby
transformation of the nuclear genome is preferred, a nucleic acid which
encodes a chloroplast transit peptide is employed 5' of the nucleic acid
sequence encoding a glycolate dehydrogenase, with this transit peptide
sequence being arranged between the promoter region and the nucleic acid
encoding the glycolate dehydrogenase so as to permit expression of a
transit peptide/glycolate dehydrogenase fusion protein. The transit
peptide makes it possible to direct the glycolate dehydrogenase into the
plastids, more especially the chloroplasts, with the fusion protein being
cleaved between the transit peptide and the glycolate dehydrogenase when
the latter enters the plastid. The transit peptide may be a single
peptide, such as an EPSPS transit peptide (described in U.S. Pat. No.
5,188,642) or a transit peptide of the plant ribulose
biscarboxylase/oxygenase small subunit (RuBisCO ssu), for example the
chloroplast transit peptide derived from the ribulose-1,5-bisphosphate
carboxylase gene from Solanum tuberosum (GenBank: gene gi21562, encoding
the protein G68077, amino acids 1-58), where appropriate including a few
amino acids of the N-terminal part of the mature RuBisCO ssu (EP 189
707), or the chloroplast targeting peptide of the potato rbcS1 gene
(gi21562). A transit peptide may be the whole naturally occurring
(wild-type) transit peptide, a functional fragment thereof, a functional
mutant thereof. It can also be a chimeric transit peptide wherein at
least two transit peptides are associated to each other or wherein parts
of different transit peptides are associated to each other in a
functional manner. One example of such chimeric transit peptide comprises
a transit peptide of the sunflower RuBisCO ssu fused to the N-terminal
part of the maize RuBisCO ssu, fused to the transit peptide of the maize
RuBisCO ssu, as described in patent EP 508 909.

[0029] Alternatively, the polypeptides may be directly expressed into the
chloroplast using transformation of the chloroplast genome. Methods for
integrating nucleic acids of interest into the chloroplast genome are
known in the art, in particular methods based on the mechanism of
homologous recombination. Suitable vectors and selection systems are
known to the person skilled in the art. The coding sequences for the
polypeptides may either be transferred in individual vectors or in one
construct, where the individual open reading frames may be fused to one
or several polycistronic RNAs with ribosome binding sites added in front
of each individual open reading frame in order to allow independent
translation. An example of means and methods which can be used for such
integration into the chloroplast genome is given for example in WO
06/108830, the content of which are hereby incorporated by reference.

[0030] When the nucleic acids are directly integrated into the chloroplast
genome, a transit peptide sequence is not required. In that case, the
(Met) translation start codon may be added to the sequence encoding a
mature protein to ensure initiation of translation.

[0032] In a particular embodiment, the nucleic acid of the invention
encodes a glycolate dehydrogenase multi-subunit fusion protein which
comprises an amino acid sequence which targets said protein to the
chloroplast.

[0033] In another particular embodiment, the nucleic acid of the invention
encodes a glycolate dehydrogenase multi-subunit fusion protein which is
the fusion of bacterial glycolate dehydrogenase subunits.

[0034] In another particular embodiment, the nucleic acid of the invention
encodes a glycolate dehydrogenase multi-subunit fusion protein which is
the fusion of the three subunits encoded by the E. coli glc operon.

[0035] In another particular embodiment, the nucleic acid of the invention
encodes a glycolate dehydrogenase multi-subunit fusion protein which
comprises amino acids sequences having at least 60% sequence identity to
the sequences of SEQ ID NOs 2, 4 and 6 respectively.

[0036] In another particular embodiment, the nucleic acid of the invention
encodes a glycolate dehydrogenase multi-subunit fusion protein which
comprises polynucleotides sequences having at least 60% sequence identity
to the polynucleotides sequences of SEQ ID NOs 1, 3 and 5 respectively.

[0037] Subject-matter of the present invention also are plant cells, plant
tissues, plants and part or seed thereof, comprising one nucleic acid
encoding a glycolate dehydrogenase multi-subunit fusion protein and
expressing inside the chloroplast one polypeptide having the enzymatic
activity of glycolate dehydrogenase.

[0038] Particular embodiments of the nucleic acids introduced into the
plant cells, plant tissues, plants and part or seed thereof are mentioned
above.

[0040] The present invention also relates to transformed plants or part
thereof, which are derived by cultivating and/or crossing the above
regenerated plants, and to the seeds of the transformed plants,
characterized in that they contain a transformed plant cell according to
the invention.

[0041] In a particular embodiment of the invention, the transformed
plants, or part thereof, are selected among rice, wheat, barley, potato,
rapeseed, tobacco.

[0042] In a particular embodiment of the invention, the transgenic seeds,
and meal, oil or food obtained thereof, are from rice, wheat, barley,
rapeseed, or tobacco plants.

[0043] The present invention also relates to any products such as the meal
which are obtained by processing the plants, part thereof, or seeds of
the invention. For example, the invention encompasses grains obtained
from the processing of the seeds according to the invention, but also
meal obtained from the further processing of the seeds or the grains, as
well as any food product obtained from said meal.

[0053] The bacterial glcD, glcE and glcF cDNAs encoding for the subunits
D, E and F of the GDH were fused with a flexible linker encoding for the
(Gly4Ser)3 motif to a multi-subunit gene construct (FIG. 1).

[0054] Restriction sites indicated in FIG. 1 were introduced to allow
rearrangement of the glcD, glcE and glcF subunits and subcloning of the
multi-subunit cassettes into the bacterial and plant expression vectors.
Furthermore, the internal restriction sites (Pstl/Sa/l and Ncol/Xhol)
were introduced to facilitate exchange of the (Gly4Ser)3 linker
with other flexible linkers. In addition, the generation of two
gene-fusions cassettes will be possible by deleting the cDNA in the
middle position via the Sa/l/Xhol restriction sites.

[0055] The C-terminal His6 tag was introduced to enable the detection and
purification of the recombinant proteins. To avoid a potential
interference of the His6 tag on the enzymatic activity of the
multi-subunit GDH enzyme an enterokinase cleavage site is added upstream
of the His6 tag allowing the removal of the C-terminal tag.

EXAMPLE 2

Synthesis of the Multi-subunit Fusion Cassettes

[0056] Three multi-subunit fusion cassettes containing the three bacterial
subunit cDNAs in three different arrangements glcD-glcE-glcF,
glcE-glcF-glcD and glcF-glcD-glcE were designed and synthetic genes
encoding for the corresponding polypeptides DEFp, EFDp and FDEp,
respectively, were synthesized. Prior to synthesis, the synthetic genes
were codon-optimized for maximum expression yields according to the
Brassica napus codon usage. Furthermore, based on a genetic algorithm,
the synthetic genes were simultaneously optimized for a large set of
competing parameters, such as mRNA secondary structure, cryptic splice
sites, codon and motif repeats, and homogenous GC content.

[0057] To determine whether DEFp, EFDp and FDEp are capable of
complementing glycolate oxidase mutants of E. coli, complementation
analysis were done with the E. coli mutant JA155 which carry transposon
insertion in the glcD subunit of the glc operon and is incapable of
growing on glycolate as the sole carbon source. Overexpression of DEFp,
EFDp and FDEp in this mutant restored the growth of bacteria in medium
containing glycolate as the sole carbon source, indicating that all three
polyproteins are functional in vivo and can complement for the glcD
subunit of the active EcGO enzyme.

EXAMPLE 4

Subcloning of DEFp, EFDp and FDEp cDNAs into the Plant Expression Vectors

[0058] To evaluate the in vivo effect of the bacterial multi-subunit DEFp,
EFDp and FDEp polyprotein on GDH activity and biomass production in N.
tabacum cv. Petit Havanna SR1 plants, the cDNAs encoding DEFp, EFDp and
FDEp were inserted into a plant expression vector enabling recombinant
protein targeting to the plant cell chloroplasts. Transgene expression is
driven by the CaMV 35S promoter with duplicated enhancer region.

[0059] The synthesized DEFp cDNA was initially inserted into the pTRAkc
shuttle vector using the EcoRl and Xbal restriction sites upstream of the
CaMV 35S terminator generating the pTRA-nptll-DEFp plasmid. The pTRA
plasmid contains the scaffold attachment region of the tobacco RB7 gene
(gi3522871) and the nptll cassette of pPCV002 (Konz and Schell, 1986) for
selection of transgenic plants on kanamycin (FIG. 2). Subsequently, the
constitutive double enhanced CaMV 35S promoter, the 5' untranslated
region of the chalcone synthase gene and the chloroplast targeting
peptide sequence of the potato rbcS1 gene were amplified by PCR using as
template the pTRAkc-rbcs1-cTP plasmid. The amplified PCR fragment was
then subcloned into the pTRA-nptll-DEF using the Asci and Aatll
restriction sites.

[0060] The cloning of EFDp and FDEp cDNAs into the plant expression vector
was performed in a similar way. The three final constructs were
designated: pTRA-355-rbcs-cTP:DEFp, pTRA-35S-rbcs-cTP:EFDp and
pTRA-35S-rbcs-cTP:FDEp, respectively.

EXAMPLE 5

Tobacco Plant Transformation and Regeneration

[0061] The plant expression vectors were introduced into Agrobacterium
tumefaciens GV3101 cells using a Gene Pulser II electroporation system
(BioRad, Hercules, Calif., USA) according to the manufacturer's
instructions. To investigate the effect of the DEFp, EFDp and FDEp
accumulation in the stably transformed tobacco plants (N. tabacum cv.
Petit Havana SR1), transgenic T0 plants were generated by leaf disk
transformation with recombinant A. tumefaciens (Horsch et al., 1985)
using kanamycin as a selection marker. The generated plants were
cultivated in the glasshouse in DE73 standard soil with a 16 h natural
daylight photoperiod and 22° C. daytime/20° C. night-time
temperature.

[0062] Up to 33 transgenic T0 plants were screened for the presence
of the transgene and the recombinant protein by multiplex PCR and
immunoblot analysis, respectively. 33-48% of the tested lines produced
the DEFp, EFDp or FDEp, respectively, at the expected molecular size of
142 kDa. Seven T0 lines showing the highest level of the recombinant
proteins (0.03 to 0.09% of total soluble protein) were used to establish
the T1 generation.

EXAMPLE 6

Chloroplast Isolation and Enzymatic Assays

[0063] Intact chloroplasts are isolated using the procedure described by
Kleffmann et al., 2007. These preparations are free of contaminating
catalase and fumarase activity (>95% purity). Glycolate dehydrogenase
activities are measured as described in Lord J. M., 1972. 100 μg of
chloroplast protein extract is added to 100 μmol potassium phosphate
(pH 8.0), 0.2 μmol DCIP, 0.1 ml 1% (w/v) PMS, and 10 μmol potassium
glycolate in a final volume of 2.4 ml. At fixed time intervals,
individual assays are terminated by the addition of 0.1 ml of 12 M HCl.
After standing for 10 min, 0.5 ml of 0.1 M phenylhydrazine-HCl is added.
The mixture is allowed to stand for a further 10 min, and then the
extinction due to the formation of glyoxylate phenylhydrazone is measured
at 324 nm.

EXAMPLE 7

CO2 Release from Labeled Glycolate in Chloroplasts Extracts

[0064] 1 μCi of [1,2-14C]-glycolate (Hartmann Analytics) is added to 50
μg of chloroplast protein extract in a tightly closed 15-ml reaction
tube. Released CO2 is absorbed in a 500-μl reaction tube
containing 0.5 M NaOH attached to the inner wall of the 15-ml tube.
Samples are incubated for 5 h and the gas phase in the reaction tube is
frequently mixed with a syringe.

EXAMPLE 8

Assessment of Phenotype of Plants Expressing E. coli GDH

[0065] Growth of the transgenic plants producing the DEFp, EFDp or FDEp
recombinant protein in the chloroplast, was monitored by the leaf area
measurements according to the formula:

[0066] Transgenic tobacco T0 lines constitutively producing the DEFp,
EFDp or FDEp, respectively, showed a significant increase of the leaf
area (1.54-, 1.75- and 1.5-fold, respectively p<0.05), compared to
non-transgenic control plants (FIG. 4). In addition, transgenic plants
had more leaves than the wild type SR1 and additional small leaves
besides the huge ones.

[0067] The photosynthetic performance of the transgenic plants was
monitored via Licor LI-6400 by measurements of the apparent CO2
assimilation and compensation point. The apparent rate of the CO2
assimilation under ambient conditions was significantly enhanced in
transgenic DEFp and EFDp plants compared to that of the wild type.
Furthermore, the DEFp transgenic lines have a significant decrease
(P<0.05) of CO2 compensation points (54 p.p.m. CO2) compared
to control (64 p.p.m. CO2), indicating higher photosynthetic rates
for the DEFp T0 lines.

[0068] Enhanced biomass and the reduced photorespiration was further
confirmed in the T1 (Table 1) and T2 generation. Furthermore,
the performance of the wild type and transgenic lines grown without
fertilizer supplementation was analysed in the T1 generation. Under
these conditions, tobacco plants overexpressing the DEFp, EFDp and FDEp
polyproteins showed reduced chlorosis and higher biomass production then
the wild type control.

[0069] Taken together, these data indicate that the plants producing the
bacterial glycolate dehydrogenase polyprotein in their chloroplast have a
significantly increased of biomass and improved photosynthetic rate.